The fermentable fractions of biomass include cellulose (.beta.-1,4-linked glucose) and hemicellulose. Cellulose consists of long, covalently bonded insoluble chains of glucose which are resistant to depolymerization. Hemicellulose is a heterogeneous fraction of biomass that is composed of xylose and minor five- and six-carbon sugars. Although it is an abundant biopolymer, cellulose is highly crystalline, insoluble in water, and highly resistant to depolymerization. The complete enzymatic degradation of cellulose to glucose, probably the most desirable fermentation feedstock, may be accomplished by the synergistic action of three distinct classes of enzymes. The first class, the "endo-.beta.-1,4-glucanases" or .beta.-1,4-D-glucan 4-glucanohydrolases (EC 3.2.1.4), acts at random on soluble and insoluble .beta.-1,4-glucan substrates to brake the chains and are commonly measured by the detection of reducing groups released from carboxymethylcellulose (CMC). The second class, the "exo-.beta.-1,4-glucosidases", includes both the .beta.-1,4-D-glucan glucohydrolases (EC 3.2.1.74), which liberate D-glucose from 1,4-.beta.-D-glucans and hydrolyse cellobiose slowly, and .beta.-1,4-D-glucan cellobiohydrolase (EC 3.2.1.91) which liberate D-cellobiose from .beta.-1,4-glucans. The third class, the ".beta.-D-glucosidases" or .beta.-D-glucoside glucohydrolases (EC 3.2.1.21), act to release D-glucose units from soluble cellodextrins, especially cellobiose, and an array of aryl-glycosides.
The development of an economic process for the conversion of low-value biomass to ethanol via fermentation requires the optimization of several key steps, especially that of cellulase production. Practical utilization of cellulose by hydrolysis with cellulase to produce glucose requires large amounts of cellulase to fully depolymerize cellulose. For example, about one kilogram cellulase preparation may be used to fully digest fifty kilograms of cellulose. Economical production of cellulase is also compounded by the relatively slow growth rates of cellulase producing fungi and the long times required for cellulase induction. Therefore, improvements in or alternative cellulase production systems capable of greater productivities, higher specific activities of cellulase activity or faster growth rates than may be possible with natural fungi would significantly reduce the cost of cellulose hydrolysis and make the large-scale bioconversion of cellulosic biomass to ethanol more economical.
Highly thermostable cellulase enzymes are secreted by the cellulolytic thermophile Acidothermus cellulolyticus gen. nov., sp. nov. These are discussed in U.S. Pat. Nos. 5,275,944 and 5,110,735. This bacterium was originally isolated from decaying wood in an acidic, thermal pool at Yellowstone National Park and deposited with the American Type Culture Collection (ATCC) under collection number 43068 (Mohagheghi et al. 1986. Int. J. System. Bacteriol. 36:435-443).
The cellulase complex produced by this organism is known to contain several different cellulase enzymes with maximal activities at temperatures of 75.degree. C. to 83.degree. C. These cellulases are resistant to inhibition from cellobiose, an end product of the reactions catalyzed by cellulase. Also, the cellulases from Acidothermus cellulolyticus are active over a broad pH range centered about pH 6, and are still quite active a pH 5, the pH at which yeasts are capable of fermenting glucose to ethanol. A high molecular weight cellulase isolated from growth broths of Acidothermus cellulolyticus was found to have a molecular weight of approximately 156,600 to 203,400 daltons by SDS-PAGE. This enzyme is described by U.S. Pat. No. 5,110,735.
A novel cellulase enzyme, known as the E1 endoglucanase, also secreted by Acidothermus cellulolyticus into the growth medium, is described in detail in U.S. Pat. No. 5,275,944. This endoglucanase demonstrates a temperature optimum of 83.degree. C. and a specific activity of 40 .mu.mole glucose release from carboxymethylcellulose/min/mg protein. This E1 endoglucanase was further identified as having an isoelectric pH of 6.7 and a molecular weight of 81,000 daltons by sodium dodecyl sulfate polyacrylamide gel electrophoresis.
It has been proposed to use recombinant cellulase enzymes to either augment or replace costly fungal enzymes for cellulose degradation (Lejeune, Colson, and Eveleigh, In Biosynthesis and Biodegradation of Cellulose, C. Haigler and P. J. Weimer, Eds., Marcel-Dekker, New York, N.Y. 1991, pp. 623-672). The genes coding for Acidothermus cellulolyticus cellulases cloned into Streptomyces lividans, E. coli, Bacillus, or other microbial host organisms could provide an abundant, inexpensive source of highly active enzymes. However, in order to produce recombinant E1 endoglucanase, the gene encoding this enzyme must be available and well characterized.